Reconstituted cell-free protein synthesis using in vitro transcribed tRNAs

Abstract

Entire reconstitution of tRNAs for active protein production in a cell-free system brings flexibility into the genetic code engineering. It can also contribute to the field of cell-free synthetic biology, which aims to construct self-replicable artificial cells. Herein, we developed a system equipped only with in vitro transcribed tRNA (iVTtRNA) based on a reconstituted cell-free protein synthesis (PURE) system. The developed system, consisting of 21 iVTtRNAs without nucleotide modifications, is able to synthesize active proteins according to the redesigned genetic code. Manipulation of iVTtRNA composition in the system enabled genetic code rewriting. Introduction of modified nucleotides into specific iVTtRNAs demonstrated to be effective for both protein yield and decoding fidelity, where the production yield of DHFR reached about 40% of the reaction with native tRNA at 30°C. The developed system will prove useful for studying decoding processes, and may be employed in genetic code and protein engineering applications.

Reconstituting Natural Cell Elements in Synthetic Cells

“…It was additionally shown that PURE could function normally using solely in vitro transcribed tRNA, termed iVTtRNA. [ 95 ] By demonstrating the versatility that arises from in vitro synthesized tRNAs, this work is a good step towards being able to incorporate unnatural amino acids into the codon table by working around the problems of tRNA modifications present in natural tRNAs. An interesting feature of this work is that because the new codon table lacks most of the redundancy present in the natural codon table, there are many open codons for which to assign unnatural amino acids.…”

Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology

“…It should be noted that codon usage has a direct influence on the elongation rate and thus regulates cotranslational folding ( Yu et al, 2015 ). Theoretically, it could be useful to adjust the tRNA concentrations in a cell-free translation reaction to mimic those from the organism from which the recombinant protein is originally derived or to redesign the genetic code ( Hibi et al, 2020 ). In principle, this could greatly assist correct protein folding; however, such experiments remain difficult without a good method to prepare individual tRNAs ( Berg and Brandl, 2020 ) on a large scale.…”

Using Genetic Code Expansion for Protein Biochemical Studies

“…In a recent study, two sense codons (UCG, UCA) and the amber stop codon in the synthetic E. coli MDS42 strain were completely eliminated for GCE (Fredens et al, 2019). Various reports have utilized cell-free protein synthesis and in vitro transcribed tRNA sets to successfully eliminate or reassign rare codons to encode ncAAs as well as to swap codons for canonical amino acids (Iwane et al, 2016;Cui et al, 2017;Fujino et al, 2020;Hibi et al, 2020). These engineered strains and techniques will contribute to the observation of protein dynamics and the development of protein biochemical studies.…”